Rapid method of detection and enumeration of sulfide-producing bacteria in food products

ABSTRACT

A rapid method for detecting spoilage of a food sample, particularly a fish sample, by detecting and enumerating sulfide-producing bacteria (SPB). A growth medium containing iron and sulfur is combined with the food sample forming an incubation mixture which is incubated for a period of time. In one embodiment, a plurality of fluorescence measurements are taken during an incubation period of about 3 hours to 17 hours at 30° C. SPB are determined to be present in the sample if the fluorescence measurement initially increases and then decreases to form a fluorescence maximum (peak). The time to detection of the fluorescence peak can be used with a correlation schedule to enumerate the SPB in the food sample. In another embodiment, a visual test can also be used to identify color changes in the incubation mixture to provide a semi-quantitative enumeration of SPB effective after about 3 hours to 17 hours of incubation.

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application is a continuation-in-part of U.S. Ser.No. 10/016,854, filed Dec. 14, 2001, now U.S. Pat. No. 6,632,632, theentirety of which is hereby expressly incorporated by reference hereinin its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable

BACKGROUND

[0003] The present invention relates to rapid methods for detection andquantification of microorganisms in food products for human consumptionor use, and more particularly to rapid methods for the detection andquantification of sulfide-producing bacteria in fish products.

[0004] Among the most predominant bacteria associated with spoilage offish products, and food products in general are sulfide-producingbacteria (SPB) such as Shewanella putrefaciens. SPB are especiallyresponsible for spoilage of many kinds of foods such as seafood,including fish, fish products, mussels, mussel products, shellfish andshellfish products and other meat products such as poultry (e.g.,chicken and turkey), pork, beef, and lamb, and even dairy products suchas cheese. SPB are particularly responsible for spoilage in fresh orcooled aerobically-packed seafood. These bacteria are present inseawater and on the surface of all living fish and shellfish, and aretransferred to the flesh during catch and processing. They grow to highlevels and cause spoilage even when the fish are stored on ice (atapproximately 0-4° C.). The spoilage is mainly due to growth ofpsychrotropic bacteria, including S. putrefaciens.

[0005] Traditionally, microbial analysis of fish has been limited tototal viable counts. Indicator testing for total viable organisms andcoliforms are the most widely used tests for routine monitoring ofmicrobial contamination. However, during the last decade, it has beendiscovered that the shelf life of fish and shellfish is correlated tothe level of specific spoilage bacteria and not to the count of totalviable organisms. Spoilage is sensory detectable when the number ofsulfide producing bacteria exceeds 10⁷ colony forming units per gram(cfu/g) of fish muscle. In the case of cod from the North Atlantic, forexample, this level is reached after approximately 12 days when the fishare stored on ice, after approximately 7 days of storage at 4° C., andafter even shorter times when stored at higher temperatures. The growthof bacteria is exponential, and the shelf life of the fish can bepredicted from the number of SPB in the fish, and the growth rate ofthese bacteria at the actual storage temperature. In addition to sulfideproduction, SPB contribute to the accumulation of trimethylamine (TMA)in the fish. TMA is a primary component of unpleasant fishy odors.

[0006] Traditional agar plate methods have been developed for analysisof S. putrefaciens and other SPB in fish (Gram et al., 1987). In thosemethods, a non-specific growth medium is supplemented with thiosulfate,cysteine and ferricitrate. Bacteria that are able to produce sulfidefrom thiosulfate or cysteine appear as black colonies on the agar, dueto precipitation of ferrous sulfide. The detection is therefore directlyrelated to the spoilage property of the bacteria. This is a benefit inanalysis of spoilage bacteria, since the spoilage potential of themicrobe is more important that its identity. However, the agar platemethod has the same disadvantages as all other agar plate methods. Forinstance, it does not give the possibility for early warning and itrequires laboratory facilities, including an autoclave and techniciansskilled in sterile technique. The method is also time and laborintensive. Also, the lowest detection level is about 100 cfu/g, which isnot adequate for presence/absence tests. Furthermore, the requiredanalysis time is at least one to three days depending on the incubationtemperature. Since the analysis takes so long, this method of analysiscannot be used to make a timely decision regarding whether or not fishshould be purchased, sold, or used, or whether or not the fish can beused to produce other products. Therefore, more rapid methods ofdetection are desired to be able to expedite the decision-making processregarding how food products such as fish and shellfish which arecontaminated with sulfide-producing bacteria can be used, sold, orpurchased.

[0007] Other, more rapid, analyses based on bacterial growth with apossibility for early warning have been developed for total viablecounts, for several hygienic indicator bacteria and for certainpathogens. For example, a sample is inoculated in a growth medium whichis more or less specific for the bacterium that is going to be detectedor quantified. The medium has an indicator that is a specific substratefor the desired bacterium. The product produced by turnover of thissubstrate becomes detectable (for example by fluorescence) when thenumber of microbes reaches a certain level. Other examples areimpedimentary methods, where the indication is related to a change inthe number of charged molecules. In these methods, the number ofmicrobes in the sample is calculated indirectly based on the timerequired until detection and on the growth rate at the given conditions.However, all of these methods suffer from one or more deficiencies, andthere continues to be a need for methods which will more rapidly detectand/or quantify SPB which cause product spoilage.

SUMMARY OF THE INVENTION

[0008] The present invention comprises methods for rapidly detecting andquantifying sulfide-producing bacteria, for instance S. putrefaciens, ina sample of a food product comprising meat, dairy or fish. The foodsample is combined with a quantity of growth medium. The growth mediumpreferably comprises an iron compound (ferric citrate or ferroussulfate, for example) and organic and/or inorganic sulfur compounds(cysteine and sodium thiosulfate, for example) and forms an ironprecipitate (e.g., iron sulfide) when exposed to S. putrefaciens orother sulfide-producing bacteria. The growth medium and the sample formsan incubation mixture. The incubation mixture is incubated for apredetermined incubation period, for example from about four or fivehours to 16-18 hours.

[0009] The level of SPB in the sample is determined by using a visualdetection method to identify a color change, or by using fluorescencedetection methods which detect trends in fluorescence production of theincubation mixtures which are correlated with SPB numbers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The patent or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawings(s) will be provided by the Office uponrequest and payment of the necessary fee.

[0011]FIG. 1 is a graph depicting results for rapid enumeration of S.putrefaciens in pure cell culture, wherein sample 1 (♦) has 3.9×10⁷cfu/ml of S. putrefaciens; sample 2 (▪) has 9.7×10⁶ cfu/ml of S.putrefaciens; sample 3 (▴) has 2.4×10⁶ cfu/ml of S. putrefaciens; sample4 (x) has 4.8×10⁴ of S. putrefaciens; sample 5 (∘) has 4.8×10³ cfu/ml ofS. putrefaciens and sample 6 () has 3.8×10⁴ cfu/ml of of S.putrefaciens.

[0012]FIG. 2 is a graph showing the development of fluorescence in codsamples contaminated with S. putrefaciens, wherein sample 1 (♦) has1.4×10⁵ cfu/ml of S. putrefaciens; sample 2 (▪) has 1.4×10⁴ cfu/ml;sample 3 (▴) has 1.4×10³ cfu/ml; sample 4 () has 1.4×10² cfu/ml; andsample 5 () has 1.4×10¹ cfu/ml. The distance from the y-axis (start ofthe analysis) to the point where the fluorescence starts to increase isequal to the “time to detection” for S. putrefaciens.

[0013]FIG. 3 is a graph depicting the correlation (r²=⁻0.89, n=118)between the time of detection (x-axis) and the number ofnaturally-occuring sulfide-producing bacteria in a variety of fresh fishproducts determined by the standard method (y-axis), cultivated in IAL(Iron Agar Lyngby) broth and incubated at 30° C.

[0014]FIG. 4 is a color chart for assessing color of an incubationmixture after an incubation period.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention comprises a rapid method for quantificationof sulfide-producing microbes, such as Shewanella putrefaciens in foodproducts, including meat, or diary, and particularly in fish andshellfish. The method is based on the formation of iron sulfide by SPBin a liquid growth medium. Iron sulfide formation is detected as achange in the background fluorescence in the growth medium or as a colorchange in the medium from yellow to gray to black or as a blackening onthe surface of an intact portion of the sample.

[0016] With the present invention it is possible to detect and enumerateeven low numbers (<10⁴ cfu/g) of sulfide-producing bacteria in samplesof fresh fish (such as, but not limited to: cod (Gadus morhua), redfish(Norwegian haddock (Sebastus marinus)), salmon (Salmo salar), haddock(Melanogrammus aegifinus), coal fish (Pollachius virens) and wolf fish(Anarhichas lupus)) within, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16 or 17 hours or increments there between hours, whichis considered sufficiently rapid and timely to allow corrective actions(e.g., to prevent or suspend the processing, purchasing, or selling) inthe case of unacceptable bacterial contamination in the food product. Italso is possible to use the method as a “pass-fail” test for SPB atparticular levels, when desired. The detection level is 1-5 bacteria pergram of sample, which is at least tenfold more sensitive than thedetection level in the traditional agar plate method.

[0017] As bacteria start to grow and produce H₂S, the properties of themedia change as ferricitrate (or ferrous sulfate) is reduced and ironsulfide, FeS, is formed. When Fe³⁺ is reduced to Fe²⁺, the emissionfluorescence initially increases, then decreases due to the darkening ofthe sample mixture caused by an increase in the amount of the ironsulfide precipitate. This increase, then decrease, in fluorescence isexpressed as a curve with a peak. Samples are read in fluorescent signalunits (fsu), which is the relative intensity of the fluorescenceemission from the samples. Samples of fish products are liquified andmixed with an iron broth growth medium forming an incubation mixture,and are incubated for an incubation period. The incubation mixture maybe incubated, for example, at a temperature of 28° C.±0.5° C., 29°C.±0.5° C., 30° C.±0.5° C., 31° C.±0.5° C., 32° C.±0.5° C., 33° C.±0.5°C., 34° C.±0.5° C., or 35° C.±0.5° C.

[0018] Growth of SPB visibly alters the iron broth in two stages. In thefirst stage, the color of the iron broth changes from yellow to brightyellow (both transparent). In the second stage, iron sulfide precipitate(FeS) starts to form, causing the sample to become gray and eventuallyblack (opaque). When measured with the fluorometer, the first visiblestage (yellow) is expressed as an increase in fluorescence signal unitsand the second visible stage (gray) is expressed as a reduction offluorescence signal units. The time at which a fluorescence peak occursrepresents the “time to detection” of SPB of the sample. The quantity ofSPB is related to the concentration of FeS. When the concentration ofFeS in the incubation mixture reaches a particular level, thefluorescent light emitted is reduced and the fluorescence begins todecrease, the fluorescence peak having been achieved. Fluorescence ismeasured at regular intervals, for example, every half-hour or hour.Typically, at least three fluorescence measurements are necessary toidentify when the fluorescence peak occurs.

[0019] Briefly, the methods described herein may be carried out bymixing a “stomached” sample (or alternatively a “non-stomached” sample)of a food product with peptone water. A sample of the peptone watermixture is added to a growth medium (iron broth media) to make anincubation mixture and incubating the incubation mixture at apredetermined temperature (e.g. 30° C.). The results (SPB concentrationof the food product) are determined by (1) detecting a visible colorchange in the mixture from yellow to black, (2) manually taking a seriesof fluorescence measurements of the incubation mixture, including duringthe visible color change from yellow to black, or (3) automaticallytaking a series of fluorescence measurements during the course ofincubation of the sample. The conventional method for detection andenumeration of sulfide-producing bacteria can be performed as areference if desired.

METHODS

[0020] Iron Broth Media

[0021] Iron agar Lyngby (IAL) is a conventional growth medium used inthe determination of total viable counts (TVC) of H₂S-producingorganisms and is made by combining 2% peptone (Difco); 0.3% Lab Lemcopowder (Oxoid); 0.3% yeast extract (Difco); 0.03% ferric-citrate(Merck); 0.03% sodium thiosulfate (Merck); 0.5% NaCl (Merck); and 1.2%agar (Difco) dissolved in 1000 ml distilled water. The pH is thenadjusted to 8.2. To dissolve the ferric-citrate completely,sterilization of the medium at 121° C. for 15 minutes is required,obtaining a pH of 7.4±0.2. After sterilization, sterile-filteredL-cysteine; 0.04% (Sigma) is added.

[0022] In an alternate version of the iron broth medium, ferrous sulfate(Fe³⁺) could be substituted for ferric citrate (Fe²⁺) at the sameconcentration (0.03%). The alternate medium enables a color change fromyellow to gray which is detectable earlier due to a more prominentblackening of the medium.

[0023] For the methods of rapid detection of sulfide-producing bacteriadescribed herein, the samples are cultivated in iron broth (IAL mediaminus agar) rather than Iron Agar Lyngby. Peptone water is used fordilution of samples and is formed by dissolving 8.5 gm NaCl (Difco) and1 gm peptone (Difco) in 1000 ml of water. The peptone water is thensterilized at 121° C. for 15 min.

[0024] Preparation of Meat Sample

[0025] A sample of meat or fish (10 g) is homogenized in 30 ml ofpeptone water in a stomacher filterbag (Seward, model 400 6041/str)using a stomacher (Lab Blender 400). Required time in the stomacher isapproximately 4 minutes or until the sample is homogenized. A subsampleof 1.5 ml of the stomached sample is dispensed from the filterbag andadded to a sterile tube containing 4.5 ml of iron broth as describedfurther herein. The tube is capped and mixed. In an alternativeembodiment the food sample may be left intact and inserted into themedium. In one embodiment the food sample is a 1 cm³ cube.

[0026] As noted, elsewhere herein the sample of the food product maycomprise, for example, fish, shellfish, cheese, poultry, beef, pork,lamb, or other fish, meat or dairy products.

[0027] Fluorescence Measurement

[0028] In a preferred embodiment of the present invention, a laboratoryfluorometer is used to detect SPB. In this embodiment, a fluorometersuch as a TURNER DESIGNS TD-700 fluorometer can be used. The TD-700 hastwo optical filters: an excitation filter with a 380 nm narrow bandpass, and an emission filter with a 450 nm narrow band pass. The TD-700is single point calibrated using pure iron broth media for setting theoptimal sensitivity and range for the fluorometer. Pure iron broth mediais set to 400 fluorescence units (fsu). Fluorescence measurements inthis embodiment preferably are taken each 15-30 minutes and preferablyare incubated at 30° C. In another embodiment, fluorescence isautomatically measured using a COLIFAST CA-100.

[0029] Standard Fluorescence Curve

[0030] To prepare a standard curve for use in the fluorescence test, S.putrefaciens can be grown overnight in iron broth at 20° C. After thispre-enrichment step, serial 10-fold dilutions are made using peptonewater. A sample of the peptone water is transferred to iron broth andmixed. From each dilution of the iron broth mixture, two parallelsamples of 3 ml are transferred to two cuvettes. The samples arepreferably incubated in the open cuvettes at 30° C. Before using thefluorometer to measure fluorescence, the cuvette is capped and mixed byinversion in order to obtain a homogenous sample. The fluorescence isgenerally measured at room temperature (18° C.-20° C.).

EXAMPLES

[0031] Referring now to FIG. 1, the results for rapid enumeration of S.putrefaciens in pure cell culture cultivated in iron broth are shown.FIG. 1 shows the relationship between fluorescence and concentration ofbacteria with regard to the time of detection. When bacterialconcentrations are initially high, the “time to detection” (TTD) or“detection time” is very early in the incubation period, whereas, wheninitial concentration is low, “time to detection” is much later in theincubation period.

[0032] Referring to FIG. 2, the graph shows the development offluorescence in samples of cod contaminated with varying levels of S.putrefaciens. The samples are cultivated in iron broth and incubated at30° C. As bacteria increase in each sample, there is an increase influorescence over a period of time which is indicated by the upwardcurve in the graph. When the fluorescence begins to decrease as moreiron sulfide is formed, a peak has been formed. In FIG. 2, the codsample having about 10⁴ cfu/g of S. putrefaciens attains a fluorescencepeak (i.e. time to detection) in about 8 hours, for example.

[0033] Referring now to FIG. 3, the graph therein shows the correlationbetween the time to detection and the number of sulfide-producingbacteria in samples of several fish species. As shown in FIG. 3, thereis a strong negative correlation (high R²) between the number ofsulfide-producing bacteria in a sample, and the time necessary to detectthe bacteria in a sample of contaminated food, fish or meat, where timeto detection is defined as the time required to achieve maximum (peak)fluorescence.

[0034] Though not wishing to be constrained by theory, it is suspectedthat the increase in fluorescence can be explained by the decrease inthe concentration of Fe³⁺ in the broth due to the formation of Fe²⁺.When a specific concentration of sulfide-producing bacteria is reachedin the medium, the fluorescence decreases. This is visually observed asa conversion from yellow to a gray color in the incubation mixture whicheventually turns black due to FeS formation. The detection time (thex-axis in FIG. 3) is defined as the distance from the y-axis (start ofanalysis) to the point of maximum (peak) fluorescence.

[0035] In the invention contemplated herein, a method of detecting andenumerating SPB in a food sample, as defined elsewhere herein, includespreparing the food sample, for example a fish sample as a liquified foodsample. Fish or shellfish, for example may be selected from the groupcomprising fish (including cod, coal fish, wolf fish, red fish, haddock,and salmon), molluscs, marine animals, crustaceans, or any productderived therefrom. An incubation mixture is formed by combining theliquified food sample with a growth medium comprising an iron compoundand a sulfur compound. An iron precipitate is formed in the incubationmixture when SPB act upon the iron compound and sulfur compound in thegrowth medium. The incubation mixture is incubated at an incubationtemperature for an incubation period. The incubation temperature of themethod is preferably from about 28° C.±0.5° C. to about 35° C.±0.5° C.and more preferably about 30° C.±0.5° C. The incubation period isgenerally from about 3 to about 17 hours and may be any incremental timeperiod therein. A plurality of fluorescence measurements (usually atleast three) are taken from the incubation mixture during the incubationperiod. It is concluded that the food sample contains a particularquantity of SPB when the fluorescence measurements taken from theincubation mixture show a maximum fluorescence. The measurementsindicate a fluorescence peak in the incubation mixture during theincubation period (an increase followed by a decrease).

[0036] Enumeration of SPB by Fluorescence Detection

[0037] In an especially preferred embodiment of the invention, SPB infood, particularly fish, products are detected and enumerated usingmanual or automatic fluorescence measurement techniques.

[0038] Portions of an incubation mixture containing the stomached fishor fish (or food) product are incubated at a predetermined temperature,preferably 30° C.±0.5° C. and fluorescence measurements are takenmanually or automatically at predetermined intervals, for example, every15, 30, 45 or 60 minutes. Fluorescence measurements are continued to betaken at appropriate intervals at least until a peak (a maximum) in thelevel of fluorescence is detected. As noted earlier, the time until thefluorescence peak is attained is referred to as the “time to detection”(TTD) or “detection time”. TTD generally occurs during the phase ofincubation when the visible color of the incubation mixture changes fromyellow to black. Therefore, optimally, fluorescence measurements areespecially preferred to be taken during the yellow-to-black color changephase to ensure that the fluorescence peak (the fluorescence maximum) isdetected.

[0039] As noted previously, the results shown in FIGS. 1 and 2 indicatethat when concentrations of SPB are initially high, the TTD occurs earlyduring the incubation period, whereas when concentrations of SPB arelow, the TTD occurs much later during the incubation period.

[0040] TTD can be used to provide a quantitative enumeration of SPB inthe original sample of food or fish or fish product by comparing the TTDfor a particular product to a previously formulated correlationschedule, such as shown in FIG. 3 for a variety of fish speciesincluding cod, coal fish, wolf fish and salmon. It is well within theability of a person of ordinary skill in the art, given the teachingsherein, to formulate a correlation schedule such as FIG. 3 or Table 1which relates time to peak fluorescence (TTD) to concentration of SPB inthe product.

[0041] In one version of the invention for example, when the TTD iswithin about 10±0.5 hours, a fish sample is considered to have about 10⁴CFU/g of SPB or more (Table 1). A level of 10⁶ CFU/g is generallyconsidered to be the maximum level of SPB which can be present in a fishsample to be shipped in a fresh condition. When the TTD is within about6±0.5 hours, the fish sample is considered to have at least 10⁶ CFU/g.When the TTD is greater than 6±0.5 hours, the fish sample is consideredto have less than 10⁶ CFU/g.

[0042] Any such TTD or CFU/g threshold can be used in a “pass-fail”version of the present invention (for example, using the values in Table1). For example, if the threshold is 10⁴ CFU/g, then the fish sample“fails” (i.e., has at least 10⁴ CFU/g) if the TTD is less than about10±0.5 hours, and “passes” (i.e., has fewer than 10⁴ CFU/g) if the TTDis greater than about 10±0.5 hours. Similarly, if the threshold is 10⁶CFU/g, then the fish sample “fails” (i.e., has at least 10⁶ CFU/g) ifthe TTD is less than about 6±0.5 hours, and “passes” (i.e., has fewerthan 10⁶ CFU/g) if the TTD is greater than about 6±0.5 hours.“Pass-fail” thresholds which have more narrow TTD ranges can be easilycalculated for each testing site based on specific types of foodproducts desired to be tested and testing procedures and facilities ateach site. TABLE 1 Correlation of Detection Time (TTD) and Numbers ofSulfide-Producing Bacteria (CFU/g) Detection Time (hr) Number of SPB(CFU/g)  4 ± .5 10⁷  5 ± .5 5 × 10⁶  6 ± .5 10⁶  7 ± .5 5 × 10⁵  8 ± .510⁵  9 ± .5 5 × 10⁴ 10 ± .5 10⁴ 11 ± .5 5 × 10³ 12 ± .5 10³ 13 ± .5 10²14 ± .5 5 × 10¹

[0043] Visual Detection and Enumeration

[0044] In a preferred embodiment of the present invention,sulfide-producing bacteria are detected and semi-quantitativelyenumerated visually (using changes in visible light) rather than byfluorescence detection. In one embodiment, a sample of food or fish(stomached or intact) is combined with a sterile iron broth as describedabove to form a sample-media mixture (an incubation mixture). Theincubation mixture is then incubated at a predetermined incubationtemperature such as 30° C., or another temperature as describedelsewhere herein.

[0045] The color of the incubation mixture is assessed visually atintervals during the incubation period to determine at what time thesample changes color from yellow to black (FIG. 4). The color of theincubation mixture at any particular incubation time can be matched witha correlation schedule such as a detection chart (Table 2, for example)to make a semi-quantitative and quality determination of theconcentration of sulfide-producing bacteria in the food sample at thattime interval.

[0046] For example, using Table 2, if the incubation mixture is stillyellow after about 3-6 hours of incubation, the original sample isconsidered to have less than about 5×10⁶ CFU/gm. If the color of theincubation mixture has changed from yellow to black after 3-6 hours, theoriginal sample is considered to have at least about 5×10⁶ CFU/gmwherein the original sample is considered to have “bad” quality.

[0047] If the color of the incubation mixture is still yellow after fromabout 7-10 hours of incubation, the sample is considered to have lessthan about 5×10⁵ CFU/gm. If the color of the incubation mixture haschanged from yellow to black after from about 7-10 hours of incubation,the sample is considered to have at least about 5×10⁵ CFU/gm to 5×10⁶CFU/gm. Wherein the original sample is considered to have “marginal”quality.

[0048] If the color of the incubation mixture is still yellow after fromabout 11-12 hours of incubation, the sample is considered to have lessthan about 10³ CFU/gm. If the color of the incubation mixture haschanged from yellow to black after from about 11-12 hours of incubation,the sample is considered to have at least about 10³ to 5×10⁵ CFU/gmwherein the original sample is considered to have “good” quality.

[0049] If the color of the incubation mixture has changed from yellow toblack only after at least twelve hours of incubation, the sample isconsidered to have less than about 10³ CFU/gm wherein the originalsample is considered to have “very good” quality.

[0050] It will be understood by those of ordinary skill in the art thatwhen other conditions are used to incubate the incubation mixtures, forexample using other incubation temperatures, or using other growthmedia, the concentrations and incubation times listed in Table 2 may beadjusted and the determination of such adjustments are considered to bewell within the ability of a person of ordinary skill in the art usingstandard techniques. TABLE 2 Semi-Quantitative Enumeration ofSulfide-Producing Bacteria Approximate CFU/g for samples incubated iniron broth at 30° C. INCUBATION TIME (Hours until sample turns Black)(hr) SPB (CFU/g) 3-6 >5 × 10⁶  7-10 5 × 10⁵ − 5 × 10⁶ 11-12 10³ − 5 ×10⁵ >12 <10³

[0051] In one embodiment of the visual detection and enumeration method,a portion of the food product sample to be tested is removed intacttherefrom and is placed directly (without stomaching) into a containerwith the iron broth to form an incubation mixture. For example, for eachintact 2-gram portion or 1 c³ portion, about 7 ml of iron broth mediumis used. The medium with the intact portion is incubated in the samemanner as the visual detection method described above. The color of theincubation mixture of the intact portion is observed at intervals asdescribed elsewhere herein. The incubation time required to observe achange of the color of the medium from yellow to black is then matchedwith a correlation schedule such as the detection chart of Table 2 tomake a semi-quantitative determination or “quality” determination of theSPB in the original food sample as described above.

[0052] In this method wherein the food sample is an intact fish sample,the fish sample should have at least one surface which has been incontact with air and does not contain skin. When placed in the mediumthe sample should be covered by the medium. The vial containing theincubation mixture should be immediately placed in an incubator in anupright position. No disinfectants should have been used on the sample,before or after removal from the fish product. Fresh fish rather thansalted, smoked, or preserved fish are used preferably.

[0053] The present invention is not to be limited in scope by thespecific embodiments described herein, since such embodiments areintended as but single illustrations of one aspect of the invention andany functionally equivalent embodiments are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications areintended to fall within the scope of the appended claims.

What is claimed is:
 1. A method of detecting and/or enumeratingsulfide-producing bacteria in a fish sample, comprising: preparing thefish sample as a liquified fish sample; forming an incubation mixture bycombining the liquified fish sample with a growth medium comprising ironand sulfur, and wherein an iron precipitate is formed in the incubationmixture when sulfide-producing bacteria act upon the iron and sulfur inthe growth medium; incubating the incubation mixture at an incubationtemperature; taking a plurality of fluorescence measurements from theincubation mixture during the incubation period to detect a fluorescencepeak, the fluorescence peak comprising a fluorescence increase followedby a fluorescence decrease, the fluorescence decrease coinciding with anaccumulation of the iron precipitate in the incubation mixture, whereinthe period of time to the fluorescence peak comprises a detection time;and estimating the number of sulfide-producing bacteria in the fishsample by comparing the detection time to a correlation schedule.
 2. Themethod of claim 1 wherein in the step of incubating the incubationmixture, the incubation temperature is from about 28° C.±0.5° C. toabout 35° C.±0.5° C.
 3. The method of claim 1 wherein in the step ofincubating the incubation mixture, the incubation temperature is about30° C.±0.5° C.
 4. The method of claim 1 wherein in the step ofincubating the incubation mixture, the incubation period is from about 3hours to about 17 hours.
 5. The method of claim 1 wherein in the step offorming an incubation mixture, the iron is provided as ferric citrate orferrous sulfate.
 6. The method of claim 1 wherein in the step of formingan incubation mixture, the sulfur is provided as cysteine andthiosulfate.
 7. The method of claim 1 wherein in the step of taking aplurality of fluorescence measurements, the fluorescence measurementsare taken manually.
 8. The method of claim 1 wherein in the step oftaking a plurality of fluorescence measurements, the fluorescencemeasurements are taken automatically.
 9. The method of claim 1 whereinin the step of estimating the number of sulfide-producing bacteria, thecorrelation schedule comprises: Detection Time (hr) Number of SPB(CFU/g) 3-6 5 × 10⁶  7-10 5 × 10⁵ − 5 × 10⁶ 11-12 10³⁻ 5 × 10⁵ >12 <10³


10. A method of detecting and/or enumerating sulfide-producing bacteriain a fish sample, comprising: preparing the fish sample as a liquifiedfish sample; forming an incubation mixture by combining the liquifiedfish sample with a growth medium comprising iron and sulfur, and whereinan iron precipitate is formed in the incubation mixture whensulfide-producing bacteria act upon the iron and the sulfur in thegrowth medium; incubating the incubation mixture at an incubationtemperature for a predetermined incubation period which produces afluorescence peak when a particular number of sulfide-producing bacteriaare present; taking a plurality of fluorescence measurements from theincubation mixture during the predetermined incubation period; andconcluding that the fish sample has at least the particular number ofsulfide-producing bacteria when a fluorescence peak is attained by theend of the predetermined incubation period, and that the fish sampledoes not have the predetermined number of sulfide-producing bacteriawhen a fluorescence peak is not attained by the end of the predeterminedincubation period.
 11. The method of claim 10 wherein in the step ofincubating the incubation mixture, the incubation temperature is fromabout 28° C.±0.5° C. to about 35° C.±0.5° C.
 12. The method of claim 10wherein in the step of incubating the incubation mixture, the incubationtemperature is about 30° C.±0.5° C.
 13. The method of claim 10 whereinin the step of incubating the incubation mixture, the predeterminedincubation period is one of 3±0.5 hours, 4±0.5 hours, 5 ±0.5 hours,6±0.5 hours, 7±0.5 hours, 8±0.5 hours, 9±0.5 hours, 10±0.5 hours, 11±0.5hours, 12±0.5 hours, 13±0.5 hours and 14±0.5 hours.
 14. The method ofclaim 10 wherein in the step of incubating the incubation mixture, theparticular number of sulfide-producing bacteria is one of 10² CFU/g, 10³CFU/g, 10⁴ CFU/g, 10⁵ CFU/g, 5×10⁵ CFU/g, 10⁶ CFU/g, 10⁶ CFU/g, 10⁷CFU/g and 10⁸ CFU/g.
 15. The method of claim 10 wherein in the step offorming an incubation mixture, the iron is provided as ferric citrate orferrous sulfate.
 16. The method of claim 10 wherein in the step offorming an incubation mixture, the sulfur is provided as cysteine andthiosulfate.
 17. The method of claim 10 wherein in the step of taking aplurality of fluorescence measurements, the fluorescence measurementsare taken manually.
 18. The method of claim 10 wherein in the step oftaking a plurality of fluorescence measurements, the fluorescencemeasurements are taken automatically.
 19. A method of detecting orenumerating sulfide-producing bacteria in a fish sample, comprising:preparing the fish sample as a liquified fish sample; forming anincubation mixture by combining the liquified fish sample with a growthmedium having a color and comprising iron and sulfur, and wherein aniron precipitate is formed in the incubation mixture whensulfide-producing bacteria act upon the iron and sulfur in the growthmedium; incubating the incubation mixture at an incubation temperature;determining a time to color change at which time the color of theincubation mixture changes from yellow to black due to an accumulationof the iron precipitate in the incubation mixture; and estimating thenumber of sulfide-producing bacteria in the fish sample by comparing thetime to color change to a correlation schedule.
 20. The method of claim19 wherein in the step of incubating the incubation mixture, theincubation temperature is from about 28° C.±0.5° C. to about 35° C.±0.5°C.
 21. The method of claim 19 wherein in the step of incubating theincubation mixture, the incubation temperature is about 30° C.±0.5° C.22. The method of claim 19 wherein in the step of incubating theincubation mixture, the predetermined incubation period is one of 3±0.5hours, 4±0.5 hours, 5±0.5 hours, 6±0.5 hours, 7±0.5 hours, 8±0.5 hours,9±0.5 hours, 10±0.5 hours, 11±0.5 hours, 12±0.5 hours, 13±0.5 hours and14±0.5 hours.
 23. The method of claim 19 wherein in the step ofincubating the incubation mixture, the number of sulfide-producingbacteria is one of 10² CFU/g, 10³ CFU/g, 10⁴ CFU/g, 10⁵ CFU/g, 5×10⁵CFU/g, 10⁶ CFU/g, 5×10⁶ CFU/g, 10⁷ CFU/g and 10 ⁸ CFU/g.
 24. The methodof claim 19 wherein in the step of forming an incubation mixture, theiron is provided as ferric citrate or ferrous sulfate.
 25. The method ofclaim 19 wherein in the step of forming an incubation mixture, thesulfur is provided as cysteine and thiosulfate.
 26. The method of claim19 wherein in the step of estimating the number of sulfide-producingbacteria, the correlation schedule comprises: Detection Time (hr) Numberof SPB (CFU/g) 3-6 5 × 10⁶  7-10 5 × 10⁵ − 5 × 10⁶ 11-12 10³⁻ 5 ×10⁵ >12 <10³


27. A method of detecting and/or enumerating sulfide-producing bacteriain a food sample, comprising: providing a food sample; providing agrowth medium comprising iron and sulfur and disposing the food sampleinto the growth medium forming an incubation mixture, and wherein aniron precipitate is formed in the incubation mixture whensulfide-producing bacteria from the food sample act upon the iron andsulfur in the growth medium; incubating the incubation mixture at anincubation temperature; determining a time to color change at which timethe color of the incubation mixture changes to black due to anaccumulation of the iron precipitate in the incubation mixture; andestimating the number of sulfide-producing bacteria on the food sampleby comparing the time to color change to a correlation schedule.
 28. Themethod of claim 27 wherein in the step of providing the food sample, thefood sample is selected from the group comprising beef, lamb, pork,poultry, fish, shellfish, molluscs, marine animals, crustaceans, or anyproduct derived therefrom.
 29. The method of claim 27 wherein the foodsample is an intact food sample.
 30. The method of claim 27 wherein inthe step of incubating the incubation mixture, the incubationtemperature is from about 28° C.±0.5° C. to about 35° C.±0.5° C.
 31. Themethod of claim 27 wherein in the step of incubating the incubationmixture, the incubation temperature is about 30° C.±0.5° C.
 32. Themethod of claim 27 wherein in the step of incubating the incubationmixture, the predetermined incubation period is one of 3±0.5 hours,4±0.5 hours, 5±0.5 hours, 6±0.5 hours, 7±0.5 hours, 8±0.5 hours, 9±0.5hours, 10±0.5 hours, 11±0.5 hours, 12±0.5 hours, 13±0.5 hours and 14±0.5hours.
 33. The method of claim 27 wherein in the step of incubating theincubation mixture, the particular number of sulfide-producing bacteriais one of 10² CFU/g, 10³ CFU/g, 10⁴ CFU/g, 10⁵ CFU/g, 5×10⁵ CFU/g, 10⁶CFU/g, 5×10⁶ CFU/g, 10⁷ CFU/g and 10⁸ CFU/g.
 34. The method of claim 27wherein in the step of forming an incubation mixture, the iron isprovided as ferric citrate or ferrous sulfate.
 35. The method of claim27 wherein in the step of forming an incubation mixture, the sulfur isprovided as cysteine and thiosulfate.
 36. The method of claim 27 whereinin the step of estimating the number of sulfide-producing bacteria, thecorrelation schedule comprises: Detection Time (hr) Number of SPB(CFU/g) 3-6 5 × 10⁶  7-10 5 × 10⁵ − 5 × 10⁶ 11-12 10³⁻ 5 × 10⁵ >12 <10³


37. A method of detecting and/or enumerating sulfide-producing bacteriain a fish sample, comprising: providing a fish sample; providing agrowth medium comprising iron and sulfur and disposing the fish sampleinto the growth medium forming an incubation mixture, and wherein aniron precipitate is formed in the incubation mixture whensulfide-producing bacteria from the fish sample act upon the iron andsulfur in the growth medium; incubating the incubation mixture at anincubation temperature; determining a time to color change at which timethe color of the incubation mixture changes to black due to anaccumulation of the iron precipitate in the incubation mixture; andestimating the number of sulfide-producing bacteria on the fish sampleby comparing the time to color change to a correlation schedule.
 38. Themethod of claim 37 wherein in the step of providing the fish sample, thefish sample is selected from the group comprising freshwater fish,saltwater fish, shellfish, and molluscs, and crustaceans, or any productderived therefrom.
 39. The method of claim 37 wherein the fish sample isan intact fish sample.
 40. The method of claim 37 wherein in the step ofincubating the incubation mixture, the incubation temperature is fromabout 28° C.±0.5° C. to about 35° C.±0.5° C.
 41. The method of claim 37wherein in the step of incubating the incubation mixture, the incubationtemperature is about 30° C.±0.5° C.
 42. The method of claim 37 whereinin the step of incubating the incubation mixture, the predeterminedincubation period is one of 3±0.5 hours, 4±0.5 hours, 5±0.5 hours, 6±0.5hours, 7±0.5 hours, 8±0.5 hours, 9±0.5 hours, 10±0.5 hours, 11±0.5hours, 12±0.5 hours, 13±0.5 hours and 14±0.5 hours.
 43. The method ofclaim 37 wherein in the step of incubating the incubation mixture, thenumber of sulfide-producing bacteria is one of 10² CFU/g, 10³ CFU/g, 10⁴CFU/g, 10⁵ CFU/g, 5×10⁵ CFU/g, 10⁶ CFU/g, 5×10⁶ CFU/g, 10⁷ CFU/g and 10⁸CFU/g.
 44. The method of claim 37 wherein in the step of forming anincubation mixture, the iron is provided as ferric citrate or ferroussulfate.
 45. The method of claim 37 wherein in the step of forming anincubation mixture, the sulfur is provided as cysteine and thiosulfate.46. The method of claim 37 wherein in the step of estimating the numberof sulfide-producing bacteria, the correlation schedule comprises:Detection Time (hr) Number of SPB (CFU/g) 3-6 5 × 10⁶  7-10 5 × 10⁵ − 5× 10⁶ 11-12 10³⁻ 5 × 10⁵ >12 <10³